Pyrazinamide (PZA) is a critical first-line tuberculosis (TB) drug that plays a unique role in shortening the duration of chemotherapy. Despite its importance in TB treatment, the target of PZA in Mycobacterium tuberculosis (Mtb) has remained elusive. We recently identified a new target of PZA as the ribosomal protein S1 or RpsA, a vital protein involved in both translation and ribosome rescuing process called trans-translation involved in management of stalled ribosomes and damaged proteins and RNA during stress. We found that RpsA overexpression conferred increased PZA resistance and that a PZA-resistant M. tuberculosis clinical isolate DHM444 without the common mechanism of pncA mutations harbored an alanine deletion mutation at 438th amino acid in the C-terminus of RpsA as a new mechanism of PZA resistance. We confirmed biochemically that the active component of PZA, pyrazinoic acid (POA), bound to wild type M. tuberculosis RpsA but not the mutant RpsA and inhibited trans-translation rather than canonical translation. These findings validate RpsA as a target of PZA. However, it is not clear how alterations in RpsA in C-terminal region affect the binding of POA and tmRNA and PZA susceptibility and whether defect in components of trans-translation tmRNA (SsrA) and SmpB alters PZA susceptibility. We hypothesize that trans-translation pathway is important for M. tuberculosis persister survival and that defect in this pathway leads to increased susceptibility to PZA and various stresses and reduced persistence. To address these hypotheses, we will use a combined genetic, biochemical, structural biology and animal studies to elucidate the role of trans-translation in PZA susceptibility and persister biology. We will create mutants defective in the C-terminus of the RpsA and also in trans-translation (SsrA and SmpB) and assess their alterations in sensitivity to PZA and diverse stress conditions in persister assays. We will also evaluate their virulence and persistence phenotypes in mouse model of TB infection and their response to TB chemotherapy and PZA. In addition, we will determine the frequency of PZA- resistant clinical isolates with mutations in the newly identified PZA resistance gene rpsA. Finally we will use a structural biology approach to determine how changes in the C-terminus of RpsA affect the binding to POA and contribute to PZA resistance. The outcome of our studies will provide new insight into the mechanisms of PZA action and resistance and the role of trans-translation in persister survival and validate components of trans-translation as persister drug targets. These studies will provide useful information for design of new and more powerful drugs that target persisters for shortening the duration of TB treatment. PUBLIC HEALTH RELEVANCE: Pyrazinamide (PZA) is a critical frontline drug that kills persisters and plays an irreplaceable role in shortening TB chemotherapy but its mechanisms of action are poorly understood. This proposal aims to study the roles of a newly identified PZA target RpsA and its associated trans-translation pathway involved in stress survival in mechanisms of PZA action and resistance and persister biology. The outcome of the study will have implications for developing much needed new persister drugs that shorten the duration of TB treatment.